Project Summary Nearly half of the world?s population lives in areas where Aedes spp. mosquitos circulate, which can transmit several viruses of human health concern including dengue virus (DENV), Zika virus (ZIKV), yellow fever virus (YFV), West Nile virus (WNV), and chikungunya virus (CHIKV). There are no specific antivirals licensed for the treatment of these human pathogens and while the YFV vaccine provides hope for new vaccine development, there is still a need for the development of new or better vaccines for the rest of these viruses. These concerns came to the forefront during the 2015-2016 unprecedented epidemic of ZIKV in the Americas, when this virus was linked to cases of microcephaly and Guillain-Barr syndrome. For all viruses to replicate and spread, they must evade detection by the host?s innate immune system, in particular the type I interferon (type-I IFN) response. Previously, the Fernandez-Sesma laboratory has shown that the cGAS/STING innate immune sensing pathway can serve as a strong restriction factor for DENV, serotype-2, replication and that the NS2B3 protease complex and its components can antagonize both cGAS and STING. Additionally, work from the Rice laboratory at Rockefeller University has implicated that cGAS is an important restriction factor in controlling arbovirus replication. Furthermore, recent publications have illustrated that direct agonists of STING are able to potently restrict CHIKV replication. In this study, it is hypothesized that the cGAS/STING innate immune sensing pathway serves to restrict the replication of the specific human arboviral pathogens listed above and that these viruses have mechanisms of cGAS/STING antagonism. The primary objectives of this study are to determine the role of the cGAS/STING innate immune sensing pathway in antagonizing the replication of DENV, ZIKV, WNV, YFV, and CHIKV, and to identify novel viral antagonists of both cGAS and STING. By exogenous overexpression of viral components, Immunofluorescent microscopy, infection of primary cell systems, and type-I IFN reporter systems as well as computational modeling and siRNA knockdowns of the cGAS/STING and complementary pathways, this study will identify previously undiscovered arboviral antagonism of the cGAS/STING pathway, for the aforementioned arboviruses, and the restriction that both cGAS and STING impose upon the replication of these viruses. Information generated from this work has broad implications further challenging the dogma of investigating ssRNA viral infection through the lens of RNA pathogen associated molecular patterns (PAMPs) alone and expands that to include the study of viral antagonism of danger associated molecular patterns (DAMPs) induced during viral infection. Furthermore, this work can serve to inform the screening of virus specific antiviral compounds and aid in the design of vaccine prototypes.